The eukaryotic voltage-gated sodium channel is responsible for initiating and propagating electrical impulses in neurons and most excitable cells. They are the major targets of drugs and naturally occurring toxins that modify electrical activity and mutations of sodium channel genes have been linked to disease conditions such as congenital long QT syndrome, generalized epilepsy and muscle myotonia. Despite much progress in understanding the role of sodium channels in the human body, there remains a significant gap in our knowledge of the biophysical mechanisms that underpin sodium channel function. Very little is known about the structural rearrangements and the underlying forces that drive the gating transitions which allow the channels to open briefly in response to a change in membrane potential. Development of well-constrained structural models has been hampered both due to our inability to study the activation process in isolation and a lack of thermodynamic tools to experimentally measure molecular forces in complex proteins. Spectroscopic and functional studies with domain specific toxins have revealed that the voltage-dependent movement of domain IV in the sodium channel is slower than those of the other three domains. The central goal of this project is to test the hypothesis that asynchronous gating in voltage-gated sodium channels arises due to differences in molecular forces responsible for electromechanical coupling within each domain. This proposal is based on our recent findings that have led to the development of analytical tools to extract site-specific interaction energies n a model-independent fashion. This analysis will be combined with cysteine accessibility, voltage-clamp fluorimetry and single-channel recording studies to develop a well-constrained structurally relevant quantitative description of sodium channel gating. These studies will be conducted on an inactivation deficient mutant background to avoid any complications that arise due to overlap of the activation and inactivation process. In specific aim 1, we will develop a well-constrained kinetic model for activation of voltage-gated sodium channels. These studies are expected to reveal distinct features associated with sodium channel gating which are typically masked in the wild-type channels due to rapid entry into the inactivated state. In specific aim 2, we will determine the molecular mechanism of activation gating in eukaryotic sodium channels. These experiments will test the notion that the S6 segments grant access to the pore in these channels. Finally, in specific aim 3, we will probe the molecular basis of voltage-sensor and pore domain interactions in voltage-dependent ion channel using Generalized Interaction energy Analysis (GIA). The proposed studies are expected to shed new light on the molecular forces that underlie conformational changes during the activation of a voltage-dependent sodium channels.